Cancer is characterized by a chaotic set of disorders where each specific cancer type presents a unique problem with respect to diagnosis, prognosis and treatment. In spite of recent progress in earlier stage detection and molecular medicine, relatively simple, reliable, non-invasive and universal cancer diagnostic technology is still lacking.
Telomerase, a specialized reverse transcriptase, has a salient role in the process of immortalization and tumorigenesis, and is therefore considered to be a hallmark for the presence of cancer. During the cell cycle progression of a normal cell, DNA polymerase completely duplicates the genomic DNA except on the very ends of the Chromosomes—the telomeres. As a consequence, the telomeric end shortens an average of 50-200 base pairs each time the cell divides. Because of this “telomere shortening phenomenon,” normal diploid cells are “mortal” and have limited capacity to proliferate. Immortalized cells, on the other hand, overcome this obstacle through the use of special mechanisms which perform telomere maintenance.
In most tumors, the maintenance of telomeres is achieved through the expression of telomerase, which stabilizes and elongates telomeres by the de novo synthesis of telomeric DNA (Ref. 1). Telomerase activity has been identified in ˜90% of human tumors but is absent in the majority of normal tissues. A high level of telomerase activity correlates with the degree of malignancy and the likelihood of tumor progression. Furthermore, there is accumulating evidence that telomerase functions as both a DNA repair and an anti-apoptotic enzyme in addition to its role in telomere maintenance. Together these findings validate the view that telomerase is an attractive target for therapeutic intervention, diagnosis and disease monitoring.
Present methods for the detection of telomerase activity are based on variations of the two-step PCR-based telomeric repeat amplification protocol (TRAP) assay. In this assay active telomerase from cell or tissue extracts adds telomeric repeats (TTAGGG) onto the 3′ end of a synthetic telomerase substrate primer. This primer is then amplified using PCR. The PCR-amplified “products” are then detected on a polyacrylamide gel or measured by ELISA. The TRAP assay, however, has not been adopted as a routine clinical test because of its limitations: the material must be processed fresh due to enzyme lability; The assay requires the use of several different instruments and takes several hours to complete, making it an unattractive routine test; there is a high possibility that false positive results can be generated from unspecific amplification of mismatch products by PCR; and inhibition of the assay by PCR inhibitors can produce false negative results. These shortcomings presently limit the clinical applicability of the current telomerase activity detection methods.
Additionally, there remains a need for a nonisotopic detection system which results in generation of a signal comprising fluorescence emission of high intensity, can result in signal amplification, is not limited as to the chemical nature of the target molecule to be detected (e.g., versus detection of nucleic acid molecules only), can be used to bind molecular probes of various types (affinity molecules, oligonucleotides, nucleobases, and the like), and can result in simultaneous detection of more than one type of target molecule by utilizing a class of nonisotopic molecules that may be excited with a single excitation light source and with resultant fluorescence emissions with discrete fluorescence peaks that can be spectrally distinguished from each other (e.g., using detection means for fluorescence that is standard in the art).